Folia Morphol. Vol. 73, No. 4, pp. 439–448 DOI: 10.5603/FM.2014.0066 O R I G I N A L A R T I C L E Copyright © 2014 Via Medica ISSN 0015–5659 www.fm.viamedica.pl

The stratigraphical organisation of the micro­vascular systems of the porcine vocal folds H. Reinhard, A. Lang, H. Gasse

Institute of Anatomy, University of Veterinary Medicine Hannover, Germany

[Received 27 February 2014; Accepted 14 April 2014]

The cranial and caudal vocal folds (CraF, CauF) of the glottis of adult minipigs (11–27 months; n = 12) were examined after immunohistochemical application of polyclonal anti-von-Willebrand-Factor and anti-Smooth-Muscle-Actin in serial paraffin sections. This examination aimed at a stratigraphical analysis of micro- vessels; data were compared with findings in humans which had been reported in the literature. (1) The distribution of the microvessels was very heterogeneous in the CraF and in the CauF, but a common pattern existed in both. (2) Characteristic vascular zones and rows were detected; each of them displayed a specific distribution and density of blood , , , lymphatic capillaries, and lymphatic precollectors. (3) A striking feature was the presence of a subepithelial Avascu- lar Band and of a focal Avascular Area within the lamina propria of the fold’s crests. (4) The vascular zones, the rows, the Avascular Band, and the Avascular Area could be allocated to specific layers of the lamina propria: subepithelial, superficial, intermediate, deep layer. (5) The loose Avascular Area at the level of the superficial layer of the lamina propria (in both CraF and CauF) corresponded to Reinke’s space in humans in terms of structure and location. (6) The direction/ /course of blood and lymphatic microvessels shared common features with that of the human vocal fold. (Folia Morphol 2014; 73, 4: 439–448)

Key words: anti-von-Willebrand-Factor, anti-Smooth-Muscle-Actin, lymphatics, paraffin sections

INTRODUCTION trol the viscosity of the vocal fold’s [6]. In terms The viscoelastic properties of the vocal folds have of this, the mechanisms of inflow, binding (stasis), a major impact on the vibratory movement during and outflow of fluids deserve major attention. In this phonation [9]. These properties are related to se- paper, stress shall be put on the structural component veral features: Firstly, they depend on the amount of these systems, i.e. blood capillaries and lymphatic and distribution of collagenous and elastic fibres, capillaries/lymphatic precollectors. The description of which characterise the stratigraphical organisation their local distribution shall complement the detailed of the lamina propria [3, 11, 49]; secondly, they are stratigraphical examinations of the minipig’s vocal influenced by the local distribution of the amorphous folds [17], whose lamina propria is subdivided into Ground Substance [26]. 4 layers: subepithelial, superficial, intermediate, and The interstitial fluid in the Ground Substance and, deep layer. These layers are characterised by different in particular, the molecules in it affect and often con- widths, fibre qualities, and fibre densities.

Address for correspondence: Dr H. Gasse, Institute of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany, tel: 0511 8567573, fax: 0511 856827214, e-mail: [email protected]

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In particular, band-like or focal regions, which were conspicuous because of their sparse number of fibres [17] and blood vessels, attracted special attention, as they may be related to Reinke’s space in the human glottis. In humans, Reinke’s space may become frequently affected by pathological condi- tions, especially by Reinke’s oedema [15, 32, 33, 35], which influences the voice [8]. In the human vocal fold, the vascular system [16, 34, 18–20, 44–47] and the blood vascular system [23, 25, 34] have been studied intensively. However, Figure 1. Bisected porcine larynx; a — cranial fold; b — caudal topographical and stratigraphical relations have not fold; the opening of the laryngeal ventricle lying in-between; been fully considered. Consequently, the location of c — position of the vestibular ligament. the different microvascular networks in situ and their attribution to certain histological layers of the lamina propria still have to be evaluated. This is even more (saturated aqueous picric acid, filtered acid-free form- relevant because of the species-specific anatomical aldehyde 37%, acetic acid 98%; 25:10:1) at room characteristic of the pig’s glottis. A distinction between temperature for 48 h. For histological processing, the its phoniatrically relevant cranial and caudal fold (for larynx was bisected in the median plane. Then, the details see [17]) is mandatory, especially if the minipig glottis was excised from each half and cut transver- is intended to be used as a model for humans. sely at its midportion (Fig. 1). These specimens were A lightmicroscopical approach on tissue sections then treated with the standard procedure of alcoholic from the minipig was used in order to evaluate the dehydration and paraffin embedding. Serial cross topographical distribution simultaneously of both, sections (5–8 µm) were alternately stained either with blood and lymph vascular structures in situ. This was performed by the immunohistochemical application Masson’s Trichrome or haematoxylin and eosin. Four of polyclonal anti-von-Willebrand-Factor (anti-SMA) other sections were immunostained with polyclonal and anti-Smooth-Muscle-Actin (anti-vWF). anti-vWF. Another 4 sections were immunostained with monoclonal anti-SMA Clone 1A4. One section MATERIALS AND METHODS of each immunostain was selected for the histomor- The specimens were taken from the larynges of fe- phometrical examination. All sections represented male minipigs (aged 11–27 months, n = 12) of the the midportion of the folds. Göttinger Minipig and Mini Lewe Minipig breeds. All Immunohistochemical staining: anti-vWF, anti-SMA animals were obtained and euthanased primarily for the anatomical dissection classes at the University of The paraffin cross sections were deparaffinised Veterinary Medicine Hannover. All related procedures in xylol and rehydrated in a graded alcohol series. were performed in accordance with the Guidelines of Endogenous peroxidase was inhibited using 0.6% the European Convention for the Protection of Verte- hydrogen peroxide in alcohol 80% for 30 min. Three brate Animals used for Experimental and other Scientific washes (5 min each) were performed with 0.05 M Purposes (86/609/EEC). Respecting this, an approval by trisphosphate-buffered saline with Tween TBS-T pH the Lower Saxony State Office for Consumer Protec- 7.4 between each step of the procedure, if not other- tion and Food Safety was not necessary. The animals wise stated. Antigen retrieval was performed diffe- were euthanased either by injection (by a veterinarian) of rently: the sections designed to be immunostained 1.5 mL/10 kg Euthadorm® (pentobarbital sodium) or with anti-vWF were pretreated with proteinase K by captive bolt stunning and consecutive bleeding; they (Sigma-Aldrich) in phosphate-buffered saline 1:200 showed no signs of larynx-associated diseases upon at 37°C for 25 min; the sections designed to be im- examination. munostained with anti-SMA were placed in a water bath containing citric buffer (pH 6.0) and heated at Histological procedures 95–99°C for 20 min. In order to prevent non-specific The larynges were excised immediately after binding of antibodies, all sections were treated with euthanasia and immersion-fixed in Bouin’s solution normal goat serum at room temperature for 20 min.

440 H. Reinhard et al., Microvascular systems of the porcine vocal folds

A B

Figure 2. Immunohistochemical staining of two adjacent serial paraffin cross sections of the porcine glottis; A. Anti-von-Willebrand-Factor detected of blood vessels (arrowheads) and endothelium of initial lymphatics (arrows); B. Anti-Smooth-Muscle-Actin (anti-SMA) detected blood vessels (arrowheads), but no initial lymphatics (arrows: anti-SMA negative lymph ).

Table 1. Immunoreactivity of anti-von-Willebrand-Factor (anti-vWF) and anti-Smooth-Muscle-Actin (anti-SMA) in vascular cells, as recorded in the literature

Blood vascular endothelium Lymph vascular endothelium Smooth muscle cells Anti-vWF [13, 28, 30] + + – – Anti-SMA [27, 28, 38, 39] – – + +

Primary antibodies were applied for 16–18 h at 4–8°C Histomorphometric examination of the microvessels at a dilution of 1:400 (anti-vWF; Dako, Hamburg, The stained cross sections were digitised (‘Scan Germany) or at a dilution of 1:100 (anti-SMA Clone Scope’ Aperio) and subsequently histomorphometri- 1A4; Dako, Hamburg, Germany). The sections were cally examined with the aid of the graphics editing then incubated with Dako EnVision + System/HRP program Adobe Photoshop CS 3 Extended 10.0.1 (a dextran polymer with secondary antibody and (Adobe Systems). In a preliminary study [31], a com- horseradish peroxidase; Dako, Hamburg, Germany) mon pattern of vascular distribution had been found for 30 min. The reaction was visualised by exposure for both the cranial and caudal vocal folds (CraF, to diaminobenzidine in a substrate buffer (Dako, CauF). This pattern was used to make a stratigraphical Hamburg, Germany). Afterwards, the sections were subdivision of the examined tissue areas. The key fea- washed 3 times (5 min) with phosphate buffered tures of this common pattern, as shown in Figure 3, saline PBS pH 7.2, and with running tap water for were: A subepithelial Zone A, subdivided in Row 1 10 min. Appropriate negative controls by omitting and Row 2; a subsequent Zone B; and an additional the antibody were run concurrently for each used Zone C (in the CraF only). antibody. Furthermore, the CraF and the CauF were separa- ted into 4 regions: cranial side, caudal side, crest, and Distinction between blood vessels and initial ventricular fundus (Fig. 3). This separation was achie- lymphatics: anti-vWF, anti-SMA ved as follows: The CraF and the CauF were defined Different types of blood vessels, and initial lymp- as the parts which extended from the laryngeal wall hatics, were distinguished by comparing adjacent into the lumen. An imaginary ‘orientation line’ was serial sections which had been exposed to either anti- drawn from the cranial surface of the CraF’s base to -vWF or anti-SMA; the procedure of distinction was the caudal surface of the CauF’s base (red, dashed line based on the expression of vWF and smooth muscular in Fig. 3); the ventricular fundus was located underne- alpha-actin in different cells (Fig. 2), as reported in ath this line. Then another line (i.e. blue lines in Fig. 3) the literature (Table 1). was placed half way between the orientation line

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Figure 3. Graphical representation of a cross section of a porcine glottis with its cranial fold, caudal fold, and ventricle (CraF, CauF). The regional separation (cranial side, crest, caudal side, fundus) and the stratigraphical subdivision (Zone A, B, C) are shown. Inset: histological representation of the caudal part of the CauF with marked Zone A, subdivided into Row 1 and Row 2, and Zone B. Paraffin section, anti-von- -Willebrand-Factor.

Figure 4. Cross section of a porcine glottis with its cranial fold (CraF), caudal fold (CauF), and ventricle. The common distribution pattern (Zones A, B, C) and the distinct Avascular Areas (blue dashed lines) are illustrated. Inset: magnified image of the caudal part of the CauF with marked Zone A, subdivided into Row 1 and Row 2, and Zone B. Paraffin section, anti-von-Willebrand-Factor.

and the highest point of the fold; this line separated included in the measurements. The mean values of the ‘crest’ from the ‘cranial’ and ‘caudal’ side of each every minipig were calculated and listed — for every fold. The placement of these division lines had to be zone, and every row of the different regions. From specially adapted for each glottis of the different these individual mean values, a common mean value animals because of the large variability in the fold’s of all minipigs of the same age group was calculated shape and size; (e.g., the CauF often appeared bent — for every zone, and every row of the different re- in the cranial direction). gions. These mean values were taken as a descriptive The type of blood vessels was distinguished on the equivalent of the vascular density within the different basis of their calibres, i.e. blood vessels smaller than regions of the folds. 10 µm were designated as blood capillaries, while blood vessels larger than 10 µm were designated as RESULTS arterioles, and venules. The lymphatic vessels — i.e. Common distribution pattern lymphatic capillaries and precollectors — were refer- red to as ‘initial lymphatics’. The distribution of the microvessels was very he- In every zone and row of the 4 regions, 10 ran- terogeneous in the CraF and in CauF, but basically, domly selected distances between the profiles of a common pattern existed in both (Fig. 4): A highly va- blood capillaries, arterioles and venules, and initial scularised, 100–300 µm wide ‘Zone A’ was located near lymphatics were measured. If fewer than 11 vascular the epithelium. This zone comprised vascular profiles profiles were encountered in a region, these were all arranged in two rows: ‘Row 1’ consisted of blood ca-

442 H. Reinhard et al., Microvascular systems of the porcine vocal folds

Figure 5. Graphical representation of a cross section of a porcine glottis with its cranial fold (CraF), caudal fold (CauF), and ventricle, to display distances between vascular profiles, and vascular diameters. The folds and the ventricle are covered by the epithelium (*); there is a distinct Avascular Band (**) underneath. The distances between the vascular profiles (coloured numbers without parentheses) and the dia- meters of the vascular profiles (coloured number in parentheses) are displayed. The colour code: blood capillaries (red), arterioles and venules (blue), lymphatics (green). Note the distinct Avascular Area (brown, dashed lines) in CraF and CauF. Note also that in Zone B the lymphatics occurred only sporadically; the percentage (i.e. 75%, 25%, 40%) indicates in how many of the minipigs lymphatics were found.

pillaries, and ‘Row 2’ consisted of arterioles and venules crest (Fig. 5). The data indicate that the folds’ crests (see inset in Fig. 4). Lymph capillaries and precollectors were supplied by capillary networks of relatively low were almost exclusively distributed within Row 2. densities (due to wide distances between profiles); ‘Zone B’ — approx. 1200 µm wide in the CraF, this was most distinct in the crest of the CauF. The 200–550 µm wide in the CauF — displayed a homo- capillaries’ diameters, i.e. the widths of their profiles, genous distribution of blood capillaries mixed with were quite uniform (7–9 µm) in all locations. arterioles, venules, and sporadic lymph capillaries Between the epithelium and the blood capillaries or precollectors. An additional, deep ‘Zone C’ was of Row 1, there was always a distinct ‘Avascular Band’ a special feature of the CraF (Fig. 4). This zone was (Fig. 5**, Fig. 6). It was widest at the CraF’s and CauF’s poorly vascularised, with only a few blood vascular crests. The width of this Avascular Band was defined as profiles with larger diameters. the distances between epithelium and capillary profi- At the crest of CraF and CauF, there was a distinct les. These distances measured 14–22 µm (Fig. 6), but ‘Avascular Area’ (blue dashed lines in Fig. 4) located a few exceptions existed as some blood capillaries were at the transition between Zone A and Zone B. located as close as 5–9 µm underneath the epithelium. The common features are graphically displayed in Row 1 was 37 µm (minimum) to 61 µm (maxi- Figure 4. Further findings shall now be described for mum) wide, depending on the location (Fig. 6). each zone, and row in greater detail; Figures 5 and 6 Row 2 (Fig. 5) contained arterioles and venules. illustrate this complex description. The distances between these vascular profiles were much larger than those in Row 1 (i.e. 132–285 µm), Zone A but — as in Row 1 — they also displayed some to- In Zone A, the superficially located Row 1 (Fig. 5) pographical differences (Fig. 5). Firstly, in the CauF, was characterised by capillaries. The arrangement of the profiles were much closer to each other than in the capillary profiles within this row was relatively the CraF; in other words, the vascular network of dense. However, the distances between them varied the CauF appeared to be denser than in the CraF. Se- with location; for instance: 37 µm (mean value) at the condly, the area of the crests of both folds displayed CraF’s caudal side vs. 55 µm (mean value) at the CraF’s relatively large distances between profiles (indicating

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Figure 6. Diagram to display the distances (minimum and maximum) between epithelium and blood capillaries (Row 1), arterioles, venules (Row 2), and lymphatics; the width of these rows (mean values) are written in the coloured columns. The Avascular Band underneath the epithelium (see Fig. 5**) is indicated in light grey; CraF — cranial fold; CauF — caudal fold.

low vascular density). However, at the cranial sides In most regions of the vocal folds, the lymphatics of both folds, the distances were even larger, and — were cut transversally. Nonetheless, on the CraF’s moreover — in the ventricular fundus, the greatest caudal side, and in the ventricular fundus, and on distances of all were encountered (Fig. 5). the CauF’s cranial side, most of them were cut lon- The diameters of the arterioles and venules were gitudinally. 16–48 µm; the largest were found in the ventricular Within Row 2, the lymphatic profiles were found fundus (Fig. 5). The profiles of the vessels displayed in an area which was 47–137 µm wide (Fig. 6). The a circular or oval shape; the distribution of both distances between the lymphatics and the epithelium types — circular or oval — was homogenous. These were quite variable; they ranged from a minimum features indicate that in most regions of the cross of 35–83 µm to a maximum of 90–195 µm (Fig. 6). sections of the vocal folds, the arterioles and venules Special attention should be given to a distinct were cut transversally; only in the ventricular fundus Avascular Area (brown dashed lines in Fig. 5), which were some of them cut longitudinally. did not contain any microvessels at all. In the CraF, The vascular profiles of the arterioles and venules this Avascular Area was larger than in the CauF: 900 — Row 2 — were 35–63 µm away from the epithelium × 250 µm (0.2 mm2) vs. 460 × 100 µm (0.04 mm2); in (Fig. 6). Row 2 was much wider than Row 1: Its width the CraF, it was located further away from the epithe- was 53 µm at a minimum, 104 µm at a maximum, lium when compared to the CauF: 100 µm vs. 40 µm. depending on the location (Fig. 6). Zone B The Lymphatics, i.e. lymph capillaries and pre- collectors, were almost exclusively arranged within Zone B (Fig. 5) was characterised by blood capil- Row 2. In most regions, the distances between these laries, arterioles, venules, and lymph capillaries or vascular profiles were greater than those between the precollectors. The arrangement of the blood vascular arterioles and venules (i.e. 151–254 µm), although the profiles was very homogenous, whereas the lympha- density of the lymphatics varied with location, too tics were found only sporadically. Particularly in the (Fig. 4): In the CauF, the density of the lymphatic pro- ventricular fundus, lymphatics were detected only in files was relatively high, particularly at its cranial side. 25% of the examined minipigs (Fig. 5). Altogether, the The diameters of the lymphatics ranged between vascular network of Zone B was relatively wide when 13 µm and 52 µm; the largest diameters of the lymp- compared with Zone A; for instance, the distance hatic profiles were found in the ventricular fundus, i.e. between the blood capillaries was 111 µm (mean in the same region that contained the largest profiles value) in the CraF, and 95 µm (mean value) in the of the arterioles and venules (Fig. 5). CauF (Fig. 5). However, there were some differences

444 H. Reinhard et al., Microvascular systems of the porcine vocal folds

with respect to the location (Fig. 5): Within the CauF, neous detection of the blood-vascular and lymphatic- the profiles of all types of vessels were much closer -vascular systems in the same tissue sample. Other to each other than in the CraF. conventional, established methods such as vascular The diameters of the vascular profiles increased corrosion casts were not used because they are tech- towards deeper parts of the lamina propria; only the nically restricted to display only the one or the other diameters of the blood capillaries remained constant vascular system, but not both at the same time. How­ (Fig. 5). In Zone B, again, it was the area of the ventricu- ever, as the detection of their topographical relation lar fundus that contained profiles with the largest dia- and of their stratigraphical arrangement in situ (i.e. in meters: 16–91 µm (arterioles, venules) and 57–73 µm the tissue section) was a primary aim of this study, the (lymphatics). The distribution of the circular and oval Corrosion-cast method did not appear appropriate. profiles in the folds’ cross sections was homogenous; Distinction between blood vessels and most microvessels, however, were cut transversally. initial lymphatics: anti-vWF, anti-SMA The distance between Zone B and the epithelium was 240 µm in the CraF and 270 µm in the CauF To date, a variety of specific lymphendothelial (measured from the highest point of the crest). The markers (Prox-1, D2-40, Lyve-1), which are exclu- width of Zone B differed markedly: In the CraF, sively present on lymphatic endothelial cells, have Zone B was quite wide (1200 µm), while in the CauF been proposed to distinguish between blood and it was only half as wide (560 µm). lymphatic vessels [40]. These markers: Prox-1 [22, 41, 48], D2-40 [4, 14, 36], and Lyve-1 [2, 5] have been Zone C reported to specifically stain lymphatic endothelial Zone C occurred only in the CraF and was located cells in different species. However, in our preliminary very deep, i.e. 1440 µm away from the epithelium. studies (unpublished data), none of these commer- This was at a proportional level which was, in the cially available lymphendothelial markers (polyclonal CauF, occupied by the thyroarytenoid muscle. Zone C Prox-1, polyclonal Lyve-1, monoclonal D2-40) yielded contained homogenously distributed blood capilla- reliable results in several tested tissues of different ries, arterioles and venules. However, no lymphatics organs of the minipig, despite various modifications were detected. The distances between the vascular in the laboratory protocols. profiles were much greater than those of the CraF’s Instead, anti-vWF and anti-SMA were highly effi- and CauF’s Zone B. The diameters of the arterioles and cient for detecting blood and lymph vascular structu- venules (21–69 µm) were much larger, too (Fig. 5). res and were suitable — by combining of the results of the two procedures — for describing the vascular DISCUSSION system adequately. The vWF is a multimeric glycoprotein of approxi- Animals mately 220 kDa polypeptide units. In the cytoplasm Only female minipigs were included in this exami- of endothelial cells, vWF is stored in Weibel-Palade nation, because at this point of the study, we wan- bodies [21, 24, 29]. The vWF is a marker of blood ted to discriminate the sexual dimorphism and the vascular and lymphatic endothelium and is therefore probably related histological differences. The phy- regarded as a panendothelial marker. The lymphatic siological characteristics of the two breeds Göttinger endothelium shows a weaker intensity and more focal Minipig [37] and Mini-Lewe Minipig [7] appeared staining when compared with blood vascular endo- to be similar enough not to require a consideration thelium because of the sparse and attenuated cyto- of breed-related differences. The 11–27-month-old plasm of the lymphatic endothelial cells [13, 28, 30]. animals were in the process of completing skeletal Smooth muscular alpha-actin is an isoform typical development and adult weight development, and for smooth muscle cells. It is present in high amounts were sexually mature [42, 43]; accordingly, these in vascular smooth muscle cells and is also expressed animals were classified as adult. by pericytes. Lymphatic capillaries do not contain smooth muscle cells, and precollectors contain them Immunohistochemistry only sporadically. Therefore, smooth muscle alpha- The alternating application of the two antibodies -actin is an appropriate immunohistochemical marker — anti-vWF and anti-SMA — facilitated the simulta- for detecting blood vessels [27, 28, 38, 39], while

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lymphatic capillaries and precollectors are excluded/ The situation differed only slightly in the CauF: the /discriminated. first third of Zone B still lay within the SL, the second third within the IL, and the last third was located The measurements and the description in the DL. One reason for this was the fact that the of the vascular system CauF lacked Zone C and deeper parts of the DL. We The measurements regarding the microvessels suggest that the thyroarytenoid muscle of the CauF served only as a descriptive tool. A statistical analy- corresponds functionally and topographically to Zone C sis was not intended and did not seem appropriate. and to deeper parts of the DL of the CraF, in accor- For instance, a random test (unpublished data) had dance with previous descriptions of Lang et al. [17]. shown that in some cases the standard deviations were quite high. Therefore, the measured values were Functional considerations with used only as an element of description rather than respect to the body-cover model an element of statistical analysis. As such, the data According to the body-cover model [11], there is are valuable to describe the location and topogra­ a loose and flexible ‘cover’ consisting of the epithelium phical relation of the vascular profiles, e.g. in terms and of the superficial part of the lamina propria. In of distances from the surface epithelium, or interva- the region of the cover, the main deformation of the scular distances. vocal fold occurs [11]. In the minipig, this loose tissue layer was separated from the surface epithelium by The microvascular system as a component of the a fibro-elastic subepithelial layer, SEL [17]. This SEL was stratigraphical organisation of the vocal folds described by Lang et al. [17] as a membrane, which The measured distances between the epithelium was placed over the loose, soft parts of the vocal and the vascular profiles were correlated with the folds. In this dense SEL — almost congruent with the topographical subunits of the lamina propria which Avascular Band — microvessels were markedly sparse. have been reported by Lang et al. [17], because these However, underneath — well protected by the densely proportional subunits comprised ‘Regions of Interest fibrous SEL — the relatively dense blood-capillary ne- (ROI)’ of known widths/diameters [17]. Respecting twork of Row 1 was located within the SL. Moreover, these numerical (metrical) pre-requisites from the the Avascular Areas in the region of the folds’ crests literature, each of the characteristic Zones and Rows, were a special feature of the SL, which was very loose as well as the Avascular Band, and the Avascular Area (with only a few collagen fibres and elastic fibres), of the microvascular system of the CraF and the CauF especially in the CraF [17]. The functional impact of in our study could be allocated to one of these specific this finding is discussed below (see: Reinke’s space). layers of the lamina propria: subepithelial, superficial, Underneath the ‘cover’, the ‘body’ comprised the intermediate, deep layer (SEL, SL, IL, DL; described deeper parts of the lamina propria, and — in the por- in the study of Lang et al. [17]): In both vocal folds, cine CauF (as in the human vocal fold) — the vocalis the Avascular Band was almost congruent with the muscle; it remains rigid during vocal fold vibration SEL, which Lang et al. [17] had characterised by high [10, 49]. In the CauF, the deep third of Zone B (ad- amounts of fibres per area. Within the underlying SL jacent to the vocalis muscle) characterised the body (a loose region of only few collagen fibres and elastic as homogenous, relatively densely vascularised, and fibres), the Avascular Area, and Row 1, and Row 2 supplied with blood capillaries, arterioles, venules, were located together with the initial lymphatics (all and sporadic initial lymphatics. The same was true of them components of Zone A). Moreover, in the for the CraF, but — due to the lack of a muscle — the CraF’s crest, the Avascular Area was topographically fibrous part of the body reached further down and almost identical with the SL of Lang et al. [17]. comprised the poorly vascularised Zone C. Furthermore, in the CraF, the Zone B (with a ho- mogenous distribution of blood capillaries mixed with Reinke’s space arterioles, venules, and sporadic initial lymphatics) Areas of low fibre density and with large amounts corresponded to the IL, while Zone C (poorly vascu- of interstitial amorphous Ground Substance in the larised) was almost congruent with the DL, which human vocal fold had been related with clinical symp- was described by Lang et al. [17] as filled with large toms of Reinke’s oedema [15, 32, 33, 35]. These loose amounts of thick collagen fibre bundles. tissue areas were referred to as the superficial layer

446 H. Reinhard et al., Microvascular systems of the porcine vocal folds

of the lamina propria, SLLP, called Reinke’s space vibration during phonation [23, 25]. With regard to [12]. In the minipigs’ CraF and CauF, there was the the above mentioned structural similarities, the same above-mentioned, loose, Avascular Area at the level can be assumed also for the porcine glottis. of the superficial layer, SL, which corresponded stru- cturally to Reinke’s space in humans. However, care CONCLUSIONS should be taken to respect two distinct features; The orientation of the lymphatic vessels was quite (1) this Avascular Area had a focal rather than a layered similar to that of the blood vessels: In humans, the extension, mainly located centrally in the crest of the lymphatics of the free edge of the vocal fold run in the CraF, and caudally in the crest of the CauF; (2) it was direction of the fold’s long axis, while the lymphatics ‘covered’ by the dense subepithelial layer, SEL [17] — in the ventricle and in the area of the subglottis run which may act as a membrane and therefore have an perpendicular to the vocal fold’s long axis [18, 34, 44, impact on the spatial limitations of interstitial-fluid 45]. In the porcine glottis, the situation is basically expansion towards the mucosal surface. This may the same, but two peculiar findings made the system be relevant for interpreting something like Reinke’s more special: many lymphatic vessels on the CraF’s oedema in the pig. caudal side, and in the ventricular fundus, and on the CauF’s cranial side ran perpendicular to the folds’ long Species-related comparison of the axes. The direction of lymph drainage — towards the microvascular networks arytenoid region and towards the ventricular fundus The comparison of the microvascular networks in the CraF and CauF — appears to be analogous to of the glottis of pigs and humans displayed both, that in the human vocal fold [18, 34, 44, 45]. differences and similarities. The most striking difference was related to the REFERENCES densities of the blood vascular networks: in humans, 1. Alipour F, Jaiswal S (2008) Phonatory characteristics of the blood-vascular network of the vocal fold has been excised pig, sheep, and cow larynges. 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